Abstract

CheY is a 14 kDa cytoplasmic protein that is activated by the transfer of a phosphoryl moiety to Asp-57 from phosphoCheA during signal transduction in bacterial chemotaxis. It has been established that metal ions are necessary for the autophosphorylation of CheA, the transfer of phosphate from phosphoCheA to CheY and the autodephosphorylation of phosphoCheY. In this work, paramagnetic relaxation enhancement has been used in conjunction with one- and two-dimensional n.m.r. to study the interaction of CheY with bivalent metal ions. These studies have led to the discovery of two conformations of the protein in water, corresponding to the metal-free and the metal-bound states. Binding of bivalent cations like Mg2+, Ca2+, Sr2+, Zn2+ and Mn2+ results in a conformational change from the metal-free to the metal-bound state. Preliminary assignments of the aromatic proton resonances are reported. Comparison of phase-sensitive double-quantum-filtered COSY, homonuclear Hartmann-Hahn coherence transfer and nuclear Overhauser enhancement spectra from the metal-bound and metal-free protein indicates that Trp-58, Thr-87 and Tyr-106 are particularly affected by the conformational change involved, and that this change is limited to a small number of residues. In addition, homonuclear Hartmann-Hahn coherence transfer experiments with paramagnetic Mn2+ show significant suppression of cross-peaks associated with Trp-58 and several neighbouring residues. Comparison of the distances estimated using n.m.r. with the CheY crystal structure indicates that the n.m.r. results are consistent with bivalent metal binding at the cluster of aspartic acid residues that includes Asp-13 and Asp-57. These studies also demonstrate the utility of paramagnetic metal-induced relaxation in conjunction with two-dimensional n.m.r. measurements for exploring ligand-binding sites.